ABSTRACT Akeyhallmarkofhumancanceriscancer-specificmetabolicrewiring.Notably,thisareaofresearchhasgained renewedattentioninthelastdecadeafterseveralstudiesdemonstratedthatkeyoncogenesinhumancancer, such as AKT, KRas, MYC or NOTCH1, have differential and specific effects on primary cellular metabolism, leadingtothehypothesisthatselectivetargetingofthoseroutesmightbeanattractivetherapeuticapproach.In thiscontext,IrecentlyidentifiedglutaminolysisasacriticalpathwayinNOTCH1-drivenT-lineageacute lymphoblasticleukemia(T-ALL),ahematologicalmalignancywhererelapsesoccurinupto20%ofpediatric and50%ofadultpatients,whoultimatelysuccumbtorefractorydisease.Importantly,inhibitionofglutaminolysis genetically,viadeletionofglutaminase(Gls),orpharmacologically,usingGlsinhibitors,resultsinanti-leukemic effectsandishighlysynergisticwithanti-NOTCH1therapies.Notably,Glsselectiveinhibitorsarecurrentlybeing explored in clinical trials for hematological malignancies, and glutaminolysis has also been proposed as a therapeutictargetinavarietyofsolidtumors.However,theroleofglutamineinvivoisstillnotwellunderstood and Gls-deficient T-ALLs eventually progress, underscoring the need to understand the mechanisms of relapse. My preliminary data suggests that glutamine-derived carbon might feed into the TCA cycle and Gls- deficient T-ALLs might still use glutamine even in the absence of glutaminase. Moreover, my preliminary data points to a role of glutaminase in stemness. Therefore, this research proposal seeks to: 1) dissect the role of glutaminase in T-ALL in vivo and unravel the mechanisms of escape to Gls loss;? 2) address the role of glutaminolysis in stemness;? and 3) identify synthetic lethal pathways/genes with pharmacological inhibition of glutaminase or with genetic loss of glutaminase using CRIPSR/Cas9 screens in vitro and experimental therapeutic experiments in vivo. These studies will reveal as yet undiscovered fundamental mechanisms implicatedinthemetabolicandepigeneticrewiringofT-ALL,willadvanceourunderstandingoftheroleofGls andglutaminolysisincancer,andwillhelpusrationallydesigncombinationsofmetabolicorepigenetictargeted therapiesthatwillresultinstrongertherapeuticeffectswithdecreasedchancesofrelapse.